Alternative splicing variant of OATP1B3 mRNA

ABSTRACT

[PROBLEM TO BE SOLVED] 
     An object of the present invention is to provide a novel tumor marker and use thereof. In more detail, the present invention provides a novel tumor marker, a method for measuring said tumor marker and a measurement kit, a method for detecting cancer using the same, a kit for detecting cancer, a method for screening a preventive and/or therapeutic agent for cancer, as well as a medicament such as cancer vaccine. 
     [SOLUTION] 
     According to the present invention, a method for measuring an alternative splicing variant of OATP1B3 mRNA in a sample to be examined is provided. Said measurement method comprises measuring mRNA comprising a nucleotide sequence represented by SEQ ID NO: 1 in the sequence listing table in a sample to be examined isolated from living organism with differentiation from a mRNA comprising a nucleotide sequence represented by SEQ ID NO: 2. Said measurement method is useful for detecting cancer or screening a preventive and/or therapeutic agent.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of Japanese Application No.2010-035166, filed on Feb. 19, 2010, and Japanese Application No.2011-032501, filed on Feb. 17, 2011. The contents of these applicationsare hereby incorporated by reference in their entirety.

TECHNICAL FIELD

The present invention relates to a novel tumor marker. In more detail,the present invention relates to a novel alternative splicing variant ofOATP1B3 mRNA, and use thereof.

BACKGROUND ART

Along with recent advances in medical technology, technology for cancertherapy has also been improved extensively, and it can occasionally beseen that there are some types of cancers which show declining trend ofmortality rates. However, findings of cancer development in early stageinfluence on the therapeutic effects, and establishment of a methodwhich enables to detect the tumor at early stage has been desired.

As to the diagnosis of cancer, in addition to the methods which detectcancer tissues visually by ultrasound or X-ray, the method for detectingabnormality such as cell morphology in the tissue collected from apatient by immunohistological procedures, as well as biochemical,immunochemical detection methods for the dynamic state of so calledtumor marker such as specific protein or gene which may cause alterationin their expression in association with the development of a specificcancer, have been known.

In these methods, the method in which the tissue is observed for itsmorphology by an immunohistological procedure needs for preparing tissuespecimen, and takes time to ascertain the presence of cancerdevelopment, and moreover, the method will require much skill toidentify the presence of cancer development from the morphologyobserved.

Therefore, application of the method which employs the tumor markerswhich can judge the presence or absence of cancer development relativelysimply and objectively, has been often performed. However, since many ofthe tumor markers employed in the human cancer diagnosis are alsoexpressed to some extent in the normal tissue cell, and variation ofexpression of the aforementioned tumor marker has to be employed as anindex of malignant alteration in many cases, there always remainspossibility of a wrong diagnosis.

Therefore, for the purpose of detecting presence of cancer developmentobjectively and more correctly, it is desirable to use a substance as atumor marker which is not detectable in non-cancer cell, but is producedonly by cancerous cell. Taking a pancreatic cancer as an example,because there is no effective screening test method for pancreaticcancer at the present time, it may be too late for treatment at the timewhen the symptom of cancer has appeared in many cases, it would benecessary to use a specific tumor marker for pancreatic cancer to detectthe pancreatic cancer in early stage.

By the way, organic anion transporting polypeptide 1B3 (OATP1B3, geneticname: SLCO1B3) is a transporter expressed on the cell membrane showingbroad substrate recognition property, and is involved in uptake ofvarious compounds comprising anti-cancer drugs into a cell. The OATP 1B3is normally expressed specifically in hepatic cells; however, theexpression will also be found in other organs in association with thetissue becoming cancerous. Up to now, it has been reported that theexpression and the function of OATP1B3 affect patient's survival rate inbreast cancer and prostate cancer (see, Non-patent Literatures 1 and 2).In addition, in the analysis using colorectal cancer cells, it has beenreported that the expression of OATP1B3 gives the cell having resistanceagainst apoptosis (see, Non-patent Literature 3).

From the things described above, it is conceivable that the OATP1B3 maybe involved in a certain role in the cancer cell. For this reason,detection of the OATP1B3 is useful for prognostic diagnosis and efficacyevaluation of anti-cancer drug in cancer therapy, and expected to be auseful tumor marker. For example, Patent Literature 1 discloses atechnology to measure expression of LST-2 in cancer tissue from femalecolorectal cancer patients using anti-LST-2 monoclonal antibody whichrecognizes C-terminal intracellular domain of LST-2 (another name ofOATP1B3), and predict prognosis of the patient based on the measurementvalue.

However, there are many points remaining still unknown with respect toexpression and function of OATP1B3 in cancer cells, and it is presentsituation that a method of using of OATP1B3 as a tumor marker is notwell-established.

PRIOR ART LITERATURES Patent Literatures

-   Patent Literature 1: JP-A-2007-119390

Non-Patent Literatures

-   Non-patent Literature 1: Muto et al. Human liver-specific organic    anion transporter-2 is a potent prognostic factor for human breast    carcinoma. (2007) Cancer Sci. 98:1570-6;-   Non-patent Literature 2: Hamada et al. Effect of SLCO1B3 Haplotype    on Testosterone Transport and Clinical Outcome in Caucasian Patients    with Androgen-Independent Prostatic Cancer (2008) Clin. Cancer Res.    14:3312-8;-   Non-patent Literature 3: Lee et al. Overexpression of OATP1B3    confers apoptotic resistance in colon cancer. (2008) Cancer Res.    68:10315-23.

SUMMARY OF INVENTION Problem to be Solved by the Invention

In view of the conventional technology as described above, an object ofthe present invention is to provide a novel tumor marker and usethereof. In more detail, the present invention is directed to provide anovel tumor marker, a measurement method and a measurement kit for theaforementioned tumor marker, a method for detecting cancer using thesame, a kit for detecting cancer, a method for screening a preventiveand/or therapeutic agent for cancer, as well as a medicament such ascancer vaccine.

Means for Solving the Problem

With the view to the conventional technology mentioned above, theinventors of the present invention have studied intensively for thepurpose of further investigation on expression and function of OATP1B3.On the way of the investigation, the inventors have obtainedsurprisingly the following 3 findings:

(1) there exist an alternative splicing variant of the conventionallyreported OATP1B3 mRNA (herein, also referred to as “OATP1B3/wt”; “wt”means wild type);

(2) this novel alternative splicing variant is expressed specificallyand strongly in cancer cell and tissue; and

(3) from this new alternative splicing variant, some peptides orproteins can be expressed;

(herein, this alternative splicing variant is also referred to as“OATP1B3/ct”, “ct” means cancer type), and accomplished the presentinvention based on these findings.

That is, according to the 1st aspect of the present invention, there isprovided a method for measuring an alternative splicing variant ofOATP1B3 mRNA in a sample to be examined. The aforementioned measurementmethod comprises measuring a mRNA comprising a nucleotide sequencerepresented by SEQ ID NO: 1 in the sequence listing table in theaforementioned sample to be examined with differentiation from a mRNAcomprising a nucleotide sequence represented by SEQ ID NO: 2.

On this occasion, it is preferable that the OATP1B3/ct is measured usingthe presence of exon SV in the nucleotide sequence represented by SEQ IDNO: 1 as an indicator (details about “exon SV” will be mentioned later).In addition, this measurement comprises specifically amplifying theOATP1B3/ct or a partial region of cDNA thereof, for example, by anucleic acid amplification method using a primer set in the regioncontaining exon SV as one primer, and measuring the amplificationproduct. In addition, the number of nucleotide of the aforementionedprimer is preferably about 15 to about 35. Moreover, the aforementionednucleic acid amplification method is preferably RT-PCR method.

In addition, according to the 2nd aspect of the present invention, thereis provided a nucleic acid satisfying the following conditions:

(1) a nucleic acid which is capable of hybridizing with the nucleic acidcomprising a nucleotide sequence represented by SEQ ID NO: 1 under astringent condition;

(2) a nucleic acid which, when a nucleic acid comprising a nucleotidesequence represented by SEQ ID NO: 2 is coexisting, does not hybridizewith such nucleic acid under a stringent condition, or else even ifhybridized with such nucleic acid, 3′ terminal thereof serves as amismatch.

On this occasion, the aforementioned nucleic acid preferably hybridizeswith a region comprising exon SV. In addition, the number of nucleotideof the aforementioned nucleic acid is preferably about 10 or more, morepreferably about 15 to about 35. Furthermore, the aforementioned nucleicacid preferably consists of a nucleotide sequence represented by SEQ IDNO: 1 or the same nucleotide sequence as a partial region of theaforementioned nucleotide sequence, or the nucleotide sequence in which10% or less of nucleotides of these nucleotide sequences have beenreplaced. Such nucleic acid is suitably employed for the measurement ofan alternative splicing variant of OATP1B3 mRNA. On this occasion, theaforementioned nucleic acid is preferably a primer or a probe fornucleic acid amplification.

Furthermore, according to the 3rd aspect of the present invention, thereis provided a measurement kit for an alternative splicing variant ofOATP1B3 mRNA comprising the nucleic acids provided by the 2nd aspect ofthe present invention.

In addition, according to the 4th aspect of the present invention, thereis also provided a detection method of cancer. The aforementioneddetection method comprises measuring an alternative splicing variant ofOATP1B3 mRNA in a sample to be examined isolated from living organism bythe measurement method provided by the 1st aspect of the presentinvention, or by using the measurement kit provided by the 3rd aspect ofthe present invention. On this occasion, it is preferable to measure analternative splicing variant of OATP1B3 mRNA comprising the nucleotidesequence represented by SEQ ID NO: 1. Moreover, in the aforementioneddetection method, the cancer is preferably colorectal cancer orpancreatic cancer.

Furthermore, according to the 5th aspect of the present invention, thereis also provided a method for screening a preventive and/or therapeuticagent for cancer. The aforementioned screening method comprises a stepwhere an alternative splicing variant of OATP1B3 mRNA in a cultured cellobtained by culturing cancer cell in the presence of test substance ismeasured by the measurement method provided by the 1st aspect of thepresent invention, or by using the measurement kit provided by the 3rdaspect of the present invention; and a step where the obtainedmeasurement result is compared with the result in the case where theaforementioned test substance is absent and/or evaluated.

And, according to the 6th aspect of the present invention, there is alsoprovided an alternative splicing variant of OATP1B3 mRNA comprising thenucleotide sequence represented by SEQ ID NO: 1 itself. The alternativesplicing variant can be used, for example, for the screening method ofthe 5th aspect of the present invention by introducing theaforementioned variant into cells in culture, being forced expressionthereof, and producing cancelation-acquired cells in culture and thelike.

In addition, according to the 7th aspect of the present invention, thereis provided the following polypeptide:

(1) a polypeptide which comprises an amino acid sequence represented bySEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, and enhancesexpression thereof in cancer cell or cancer tissue; or

(2) a polypeptide which comprises an amino acid sequence in which 10% orless of amino acids are replaced, deleted, and/or inserted in the aminoacid sequence represented by SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,or SEQ ID NO: 11, and enhances expression thereof in cancer cell orcancer tissue. The aforementioned polypeptide is preferably apolypeptide consisting of the amino acid represented by SEQ ID NO: 5,SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11.

Furthermore, according to the 8th aspect of the present invention, thereare also provided a nucleic acid encoding the polypeptide of the 7thaspect, an expression vector comprising the aforementioned nucleic acid,and a cell transformed by the aforementioned expression vector.

In addition, according to the 9th aspect of the present invention, thereare also provided an antibody which is capable of binding to thepolypeptide of the 7th aspect, and a nucleic acid hybridizing with thenucleic acid comprising a nucleotide sequence represented by SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 under stringentcondition, having at least 15 nucleotides.

And, according to the 10th aspect of the present invention, there isprovided a detection method for cancer comprising measuring the amountof polypeptide of the aforementioned 7th aspect in a sample to beexamined isolated from living organism. On this occasion, it ispreferable to employ the antibody of the aforementioned 9th aspect forthe measurement. Also, a detection kit for cancer comprising theaforementioned antibody can be provided.

In the 11th aspect of the present invention, a medicament is provided.The aforementioned medicament comprises the polypeptide of the 7thaspect or a fragment thereof, or the nucleic acid of the 8th aspect or afragment thereof, and is the one to be used for inducing a specificcytotoxic T cell. The aforementioned medicament is preferably a cancervaccine.

Effect of the Invention

According to the present invention, a novel alternative splicing variantof human OATP1B3 mRNA is provided as a new cancer marker, and also ameasurement method for the same, a nucleic acid which can be used forthe aforementioned measurement, and a means for practicing theaforementioned measurement method (a kit for measurement) are provided.In addition, according to the present invention, a new method fordetecting cancer is also provided, and further, a new method forscreening a preventive and/or therapeutic agent for cancer is alsoprovided. In addition, there are also provided a polypeptide which canbe used for detecting cancer and an antibody binding thereto, a methodfor detecting cancer using the amount of the aforementioned polypeptideas an indicator, a kit for detecting cancer comprising theaforementioned antibody, and a medicament as a cancer vaccine and thelike.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A, FIG. 1B, and FIG. 1C, combined (“FIG. 1”), show the results ofalignment carried out for the nucleotide sequence of cDNA of OATP1B3/ct(SEQ ID NO: 1) and the nucleotide sequence of cDNA of OATP1B3/wt (SEQ IDNO: 2). In FIG. 1, an asterisk (*) means that the nucleotide isidentical between the two nucleotide sequences.

FIG. 2 is a figure showing the amino acid sequence of the proteinencoded by OATP1B3/wt.

FIG. 3 is a photograph showing the results of agarose gelelectrophoresis performed to visualize the results of RLM-5′-RACE methodcarried out using the total RNA derived from human colon cancer or thetotal RNA derived from the Caucasian liver as a template in “5.Determination of transcription starting point of SLCO1B3 gene” inExample.

FIG. 4 is a diagram showing the structure of human SLCO1B3 gene. In FIG.4, the locations of known and new transcription start sites are shown.In addition, the positions where the primer set used in the RT-PCRperformed in Example hybridize are also indicated by arrow,respectively.

FIG. 5 is a photograph showing the results of agarose gelelectrophoresis performed to visualize the results of RT-PCR carried outusing the total RNA from LS180 cell, PK45p cell, human coloncancer/normal colon pair RNA and the total RNA derived from theCaucasian liver as a template in “6. Preparation of cDNA and analysis ofmRNA expression by RT-PCR method” in Example.

FIG. 6 is a graph which shows the results (mRNA expression profiles) of“8. Analysis mRNA expression by quantitative real-time PCR” in Example.FIG. 6-A shows the expression level of OATP1B3/ct in each tissue andcell. In addition, FIG. 6-B shows the expression level of OATP1B3/wt ineach tissue and cell. Further, FIG. 6-C shows the ratios of expressionlevel (copy number of OATP1B3/ct versus copy number of OATP1B3/wt).

FIG. 7 is a figure showing four open reading frames (ORF) locating onOATP1B3/ct. The nucleotide sequence of cDNA is shown in the uppersequence, and the amino acid sequence coded thereby is shown in thelower sequence. In addition, the underline indicates ORF, and bold-facedATG indicates the estimated translation initiation codon locating on theupstream of ORF. The sign “GFP” as shown in the amino acid sequence tobe encoded indicates the site where the nucleotide sequence encodinggreen fluorescent protein (GFP) was inserted when the expression plasmidwas constructed in Example. It should be noted that, as for frame 2, theexpression vector was constructed by inserting the nucleotide sequenceencoding GFP into a position behind the region encoding N-terminal 20amino acids.

FIG. 8 is a graph showing the results of “10. Reverse transfection” inExample. In addition, green color is the fluorescence derived from theGFP; and red color is the fluorescence derived from the mCherry whichserves as a transfection control. Also, a superposition of bothfluorescences is shown as Merge, and a differential interference imageis shown as Phase.

EMBODIMENTS FOR CARRYING OUT THE INVENTION

Human SLCO1B3 is a gene of about 106 kbp locating on a chromosome 12p12,and from this gene, OATP1B3 mRNA (OATP1B3/wt; Refseq Accession No.NM_019844) which consists of 2712 bp (15 exons) is transcribed. Thenucleotide sequence of cDNA of this OATP1B3/wt which has already beenreported is represented by SEQ ID NO: 2 and FIG. 1. This OATP1B3/wtencodes a protein which consists of 702 amino acids (OATP1B3 protein;RefSeq Accession No. NP_062818). The amino acid sequence of the proteinwhich is encoded by this OATP1B3/wt is represented by SEQ ID NO: 3 andFIG. 2. In addition, in the nucleotide sequence represented by SEQ IDNO: 2, the 1st nucleotide from 5′ terminal (herein, referred to as“1nt”; the nucleotides at other positions are called similarly by addingnt to the number of position where the nucleotide is located from 5′terminal) to 61nt is exon 1; 62nt to 210nt is exon 2; 211nt to 1991nt isexon 3 to exon 14; and 1992nt to 2712nt is exon 15. In addition, thecoding sequence (CDS; Coding Sequence) of OATP1B3/wt is 127 to 2235nt(the stop codon TAA is included) over exon 2 to exon 15, and is writtenby capital letters in FIG. 1. On the other hand, the non-coding regionof OATP1B3/wt is written by lower-case letters in FIG. 1.

As described in Example mentioned later, the inventors of the presentinvention found that when the RLM-5′-RACE method was carried out for thepurpose of determining the transcription starting site of human SLCO1B3gene using the total RNA derived from human colon cancer tissue as atemplate, the amplification product with smaller molecular weight thanthe size expected from the reported information on the transcriptionstarting site (“TSS” shown in FIG. 1) was detected. And, when thenucleotide sequence nearby the novel transcription starting site of theSLCO1B3 gene was compared with the database of human genomic DNA, it wasclarified that the novel transcription starting site was located in theregion of the intron 2 in SLCO1B3 gene, and furthermore, new exonexisted in this region also (this new exons is referred to as “exonSV”). Moreover, this exon SV had been spliced to exon 3 in OATP1B3/wt(see, FIG. 4 mentioned later). The nucleotide sequence of cDNA of anovel alternative splicing variant (OATP1B3/ct) of thus discoveredOATP1B3 mRNA is represented by SEQ ID NO: 1 and FIG. 1.

As mentioned above, exon SV of OATP1B3/ct has been spliced to exon 3 ofOATP1B3/wt. That is, in the nucleotide sequence represented by SEQ IDNO: 1, the sequence from 1nt to 139nt is the exon SV. In addition, thesequence from 140nt to 1920nt in the nucleotide sequence represented bySEQ ID NO: 1 has substantially the same sequence to the sequence fromexon 3 to exon 14 of OATP1B3/wt. And, the nucleotide sequence from1921nt to 2170nt in the nucleotide sequence represented by SEQ ID NO: 1has the same sequence to the corresponding region of exon 15 ofOATP1B3/wt.

It was confirmed that an expression level of the alternative splicingvariant found in the present invention (for example, OATP1B3/ct) wasincreased in cancer cell, as described later in Example. Therefore, itis possible to detect cancer by measuring the aforementioned alternativesplicing variant in a sample to be examined isolated from the livingorganism.

Hereinafter, the specific preferable embodiments of the presentinvention will be described in detail, however, the technical scope ofthe present invention should be defined based on the descriptions ofclaims, and should not be limited by the following illustrativeembodiments and Examples.

The measurement method for alternative splicing variant of OATP1B3 mRNAprovided in the 1st aspect of the present invention comprises measuringa mRNA comprising a nucleotide sequence represented by SEQ ID NO: 1 (forexample, the mRNA consisting of the nucleotide sequence represented bySEQ ID NO: 1) in the aforementioned sample to be examined withdifferentiation from a mRNA comprising a nucleotide sequence representedby SEQ ID NO: 2 (for example, the mRNA consisting of the nucleotidesequence represented by SEQ ID NO: 2). It should be noted that, sincethe nucleotide sequence represented by SEQ ID NO:1 indicates both ofnucleotide sequences of mRNA and cDNA obtained using the same as atemplate, when mRNA is referred to, of course, since uracil (u) may becontained in place of thymine (t), “t” can be replaced by “u”. Inaddition, “measurement” encompasses any concept of quantitativemeasurement, semi-quantitative measurement and detection. Furthermore,“measurement of mRNA” encompasses, in addition to a case of measuringmRNA directly, a case where after the mRNA is once converted to cDNA,the aforementioned cDNA is measured (RT-PCR and the like as describedlater), and a case where the mRNA is measured indirectly such as thecase when translation product of the mRNA is measured. Moreover, in thefollowing description, mRNA, cDNA, nucleic acid and so on whichcomprises the nucleotide sequence represented by SEQ ID NO: 1 aresometimes referred to as “mRNA of SEQ ID NO: 1, cDNA of SEQ ID NO: 1,nucleic acid of SEQ ID NO: 1, and so on”, respectively.

In addition, in the sequence listing, even if it is double strandednucleic acid, only a single strand of them (a sense strand for thenucleic acid encoding a polypeptide) is to be indicated according to therule. For this reason, in the case of double stranded nucleic acid, evenif the sequence indicated actually in the sequence listing is a singlestrand, it can be understood that actually a strand complementary to theindicated strand is also indicated in the sequence listing. Therefore,in the present application, when the nucleic acid which comprises orconsists of the nucleotide sequence shown in a certain SEQ ID NO is ormay be double stranded nucleic acid, the nucleotide sequence shown inthe SEQ ID NO shall also include the complementary strand thereof,except for the case where it is clearly not right from the context. Forexample, “the nucleic acid which is capable of hybridizing with thenucleic acid comprising a nucleotide sequence represented by SEQ ID NO:1” means, in addition to a nucleic acid which is capable of hybridizingwith a sense strand actually represented by SEQ ID NO: 1, a nucleic acidwhich is capable of hybridizing with the antisense strand consisting ofthe nucleotide sequence complementary to the aforementioned sensestrand.

It is easy for the person skilled in the art to measure the mRNA of SEQID NO: 1 with identification from the mRNA of SEQ ID NO: 2, and ispossible by various methods. For example, by setting primers within exonSV and in the region after exon 3 to perform reverse transcriptase-PCR(RT-PCR), and differentiating whether the mRNA is OATP1B3/wt or analternative splicing variant (for example, OATP1B3/ct) based on themolecular weight of the amplification product, the mRNA can be measured.Moreover, as to the obtained amplification product, nucleotide sequencemay be determined (direct sequencing).

As mentioned above, in the mRNA of SEQ ID NO: 1, there exist exon SVwhich is an exon not existing in OATP1B3/wt. Therefore, alternativesplicing variant can be measured by using the presence of this exon SVas an indicator. That is, using the presence of exon SV in thenucleotide sequence represented by SEQ ID NO: 1 as an indicator, analternative splicing variant comprising the nucleotide sequencerepresented by SEQ ID NO: 1 can be measured.

Preferred method for measuring an alternative splicing variant by usingthe presence of exon SV as an indicator includes, a method which employsnucleic acid amplification method using a primer which is capable ofhybridizing with the region comprising exon SV, and a method using aprobe which is capable of hybridizing with the region comprising exonSV.

The method employing nucleic acid amplification method comprisesspecifically amplifying mRNA comprising a nucleotide sequencerepresented by SEQ ID NO: 1 or a partial region of cDNA thereof by anucleic acid amplification method using a primer set in the regioncontaining the aforementioned exon SV as one primer, and measuring theamplification product. In this case, since “set in the region” meanshybridizing with the region, and the nucleic acid to be amplified by thenucleic acid amplification method like PCR is double strand, asmentioned above, both of the case where the primer which hybridizes withthe sense strand described actually on the list (reverse primer) isemployed and the case where the primer which hybridizes with theantisense strand complementary to the aforementioned sense strand(forward primer) is employed are also included. Accordingly, forexample, “primer set in the region comprising exon SV” means the primerwhich hybridizes with the region comprising exon SV (that is, 1nt to139nt) of the sense strand actually described in SEQ ID NO: 1 (reverseprimer), or the primer having the nucleotide sequence complementary tothe aforementioned primer (forward primer). In addition, “specificallyamplifying” means that although the mRNA comprising the nucleotidesequence represented by SEQ ID NO: 1 or a partial region of cDNA thereofis amplified, the mRNA comprising the nucleotide sequence represented bySEQ ID NO: 2 or a partial region of cDNA thereof is not amplified. Itshould be noticed that, in the nucleotide sequence represented by SEQ IDNO: 1, 1nt to 139nt is exon SV, and exon 3 starts from 140nt. Therefore,in the nucleotide sequence represented by SEQ ID NO: 1, the fusion pointbetween 139nt and 140nt is a specific fusion point of exon SV and exon 3which does not exist in OATP1B3/wt. In this regard, however, as is clearfrom FIG. 1, two nucleotides of the upper stream side from theaforementioned specific fusion point are common to OATP1B3/wt andOATP1B3/ct. Therefore, in the nucleotide sequence represented by SEQ IDNO: 1, the fusion point between 137nt and 138nt which is the nearestother specific fusion point is more suitable as a specific fusion pointbetween exon SV and exon 3 than the fusion point between 139nt and140nt. For this reason, as “a region comprising exon SV” in the presentdescription, 1nt to 137nt is more preferable region.

The preferable example of nucleic acid amplification method foramplifying DNA having the same nucleotide sequence (note, however, thatu is replaced by t) as a partial region of mRNA to be used as a templateincludes the reverse transcriptase-PCR (RT-PCR). In the RT-PCR, mRNA isextracted from cell by the conventional procedure; using the mRNA as atemplate and by an action of reverse transcriptase, single strand cDNAis generated; further using this single strand cDNA as a templatecomplementary strand is generated, thus the double stranded cDNA isobtained. Subsequently, PCR is carried out using the obtained doublestranded cDNA as a template, the aforementioned cDNA or partial regionthereof is amplified. Since RT-PCR itself is well-known technology, anda kit and equipment thereof are also commercially available, it can becarried out easily. To amplify the cDNA derived from alternativesplicing variant specifically, the primer set in the region comprisingthe specific fusion point mentioned above is employed as one primer. Theother primer can be set in the arbitrary region of splicing variant tobe amplified. In addition, the primer size is not particularly limited,but usually about 15 to 35 nucleotides, and preferably about 15 to 25nucleotides.

For example, in the case where the mRNA of SEQ ID NO: 1 is measured bythe RT-PCR, the primer which has been set in the region comprising exonSV in the nucleotide sequence represented by SEQ ID NO: 1 is employed asone primer (it may be either forward primer or reverse primer). In thiscase, in order to amplify cDNA derived from alternative splicing variantspecifically, it is preferable to use the one as a primer whichhybridizes with the cDNA of SEQ ID NO: 1 under a stringent condition,but does not hybridize with the cDNA of SEQ ID NO: 2. Here, “a stringentcondition” is referred to a condition under which a primer specificallyhybridizes with cDNA, and for example, it may be measured according tothe condition described in a textbook (Sambrook, et al. ed., “MolecularCloning, A Laboratory Manual, 2nd ed.”, 1989, Coldspring HarborLaboratory, especially in section 11. 45 “Conditions for Hybridizationof Oligonucleotide Probes”, etc.), and is not particularly limited. Thestringent condition depends on salt concentration, temperature, andother conditions; and, for example, as salt concentration becomes lower,and temperature becomes higher, stringency becomes higher, andhybridization of primer with cDNA becomes difficult. Generally, the saltconcentration is adjusted by adjusting the concentration of SSC solution(NaCl+trisodium citrate), and the stringent salt concentration is, forexample, about 250 mM or less of NaCl and about 25 mM or less oftrisodium citrate. The stringent temperature is generally a temperaturelower by 15 to 25° C. than the melting temperature (Tm) of perfecthybridization, and, for example, about 30° C. or higher. The temperaturecan be lowered by adding organic solvent (for example, formamide) to thesolution. Other conditions include hybridization time, concentration ofdetergent (for example, SDS), and presence or absence of carrier DNA,etc.; and various stringencies can be set up by combining theseconditions. As a preferred example, hybridization is carried out underthe conditions of 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50%formamide, and 200 μg/mL of denatured salmon sperm DNA, at a temperatureof 42° C. In addition, washing condition after hybridization alsoinfluences on the stringency. This washing condition is also defined bysalt concentration and temperature, and stringency of washing increasesby decreasing salt concentration and increasing temperature. As apreferred example, washing is carried out under the condition of 15 mMNaCl, 1.5 mM trisodium citrate, and 0.1% SDS, at 68° C.

In order to ensure that the hybridization occurs with the cDNA of SEQ IDNO: 1, but does not occur with the cDNA of SEQ ID NO: 2, the primer ispreferably the one in which 20% or more thereof hybridize with theregion comprising exon SV. Alternatively, even in the case where thehybridization occurs with cDNA of SEQ ID NO: 2, it is preferable to usesuch a primer in which the 3′ terminal thereof serves as a mismatch.Even if hybridization occurred with cDNA of SEQ ID NO: 2 under astringent condition, if the 3′ terminal of the primer serves as amismatch, amplification does not take place substantially. Furthermore,it is particularly preferable to employ a primer in which at least 20%or more is capable of hybridizing with the region within exon SV underthe stringent condition, and the 3′ terminal thereof serves as amismatch to cDNA of SEQ ID NO: 2. By employing the above primer as oneprimer, even if the cDNA of SEQ ID NO: 2 is coexisting, only the cDNA ofSEQ ID NO: 1 can be amplified specifically. In addition, although aprimer which is completely complementary to the region in the nucleotidesequence of SEQ ID NO: 1 to be hybridized with the primer is preferable,usually, even if about 10% or less of mismatch exists, the primer can beused as a primer in many cases (however, as mentioned above, the onecomprising such a mismatch in the 3′ terminal of a primer should beexcluded).

The PCR in itself can be performed according to the conventionalprocedures. And, the amplification product is measured afteramplification by the PCR. Measurement of such amplification product canalso be performed by the conventional procedures. As mentioned above,the “measurement” in the present invention encompasses both detectionand quantitative measurement. Furthermore, simple determination orsemi-quantitative measurement such as measurement of fluorescenceintensity of electrophoresis band of amplification products, and visualjudgment of thickness is also included in the concept of “measurement”.The measurement of amplification products can be performed, for example,by subjecting the amplification products to electrophoresis, anddetecting the amplified bands. In addition, the amplification productcan be also measured by fluorescently labeling the amplification productby performing the PCR under the presence of fluorescently labelednucleotide triphosphate, and measuring fluorescence intensity of theamplified band. Further, the measurement of the amplification productcan be also performed by transcribing the electrophoretic pattern to themembrane consisting of nylon or nitrocellulose to hybridize a labeledprobe which is capable of hybridizing with the amplification product,and detecting or determining (PCR-Southern method) the label.Furthermore, the measurement of the amplification product can be alsoperformed by solid-phasing the probe which is capable of hybridizingwith amplification products, carrying out the above-describedamplification process in the presence of labeled nucleotide triphosphateto bind the amplification product to the solid-phased probe, andmeasuring the amplification product bound to the solid phase. All ofthese methods are conventional procedures, and well known knowledge maybe appropriately referred to.

Alternatively, preferably the PCR of the above-described RT-PCR iscarried out by real-time detection PCR, because more precisequantitative measurement of the amplification product can be performed.In the real-time detection PCR, normal PCR is carried out in thepresence of a probe bound with two kinds of fluorescent dye, which iscapable of hybridizing with the amplification product. One of the twokinds of fluorescent dyes is a quencher dye which negates fluorescentemission from another fluorescent dye, and fluorescence is not measuredin the state where two kinds of fluorescent dyes are bound to the sameprobe molecule. On the other hand, if amplification is taken place byPCR, at first, the probe will hybridize with the amplification product.And, when the probe is digested by exonuclease activity of the DNApolymerase used for the amplification reaction, the two kinds offluorescent dyes become apart, and extinction by quencher dye does notoccur, and as a consequent, fluorescence becomes measurable. Sincenumber of cycles at which the fluorescence intensity measured increaserapidly may vary depending on the concentration of template nucleic acidin a sample, the concentration of template nucleic acid in the samplecan be measured quantitatively by measuring fluorescence of reactionsolution continuously. In addition, the real-time detection PCR initself is well-known technology, and since the kit for it is alsocommercially available, it can be easily carried out employing the kitin the market.

It should be noted that, when the RT-PCR is performed, amplification maytake place sometimes using pseudogene as a template which is containedin the genomic DNA contaminated when mRNA is first extracted. Therefore,as for the primer set to be used, it is preferable that the set is theone in which the primers have been selected so as not to cause theamplification resulting from such a pseudogene. Whether theamplification resulting from a pseudogene take place or not can bedetermined, for example, by whether only the amplification product ofthe same size as the case where PCR is performed by employing cDNA as atemplate is formed or not, when PCR is performed using the primer setconcerned, and using the genomic DNA as a template. The primer sets(Table 3) exactly employed in the Example mentioned later are the oneswhich do not cause such amplification resulting from a pseudogene. Also,in some cases, before performing RT-PCR, PCR is carried out using theRNA to be used as a template, and using a primer set which amplifieshousekeeping genes, such as human glyceraldehyde-3-phosphatedehydrogenase (GAPDH) (for example, the one which is listed in Table 1),and by checking that an amplification products is not acquired, it canbe guaranteed that there is no contamination of genomic DNA.

As mentioned above, although the measurement method of the presentinvention was explained in detail by taking the case where measurementwas performed by the RT-PCR method as an example, the measurement methodof the present invention is not limited to the RT-PCR method, and anyother method utilizing the nucleic acid amplification method may beused, so long as it is the one in which mRNA of alternative splicingvariant or cDNA or partial region thereof is amplified using theaforementioned primer. For example, mRNA can also be amplified by theNASBA method (3SR method, TMA method). In the NASBA method, the reverseprimer in which T7 promoter sequence is attached to 5′ terminal thereofis used as a reverse primer in the presence of reverse transcriptasesuch as AMT-RT and the like, to form cDNA strand (antisense strand)using mRNA as a template. RNA strand of the obtained RNA/DNA hybriddouble strand is digested by RNase H to generate a single-stranded cDNA(antisense strand). Subsequently, using this single-stranded cDNA as atemplate, a complementary strand is formed using forward primer in thepresence of DNA polymerase (when AMT-RT is used as a reversetranscriptase, the AMT-RT can serve as DNA polymerase because itpossesses DNA polymerase activity), and thus double-stranded cDNA ismade up. Since this double-stranded cDNA comprises T7 promoter sequence,if T7 RNA polymerase is allowed to act on this, RNA (antisense strand)is formed successively by transcription. Furthermore, as to thedouble-stranded cDNA formed, by repeating a denaturation process, anannealing process with the above-described forward primer and thereverse primer, and an extension process of complementary strand, thedouble-stranded cDNA is amplified, and further, by the transcriptionusing this as a template, RNA (antisense strands) is also formed. Insuch NASBA method, only alternative splicing variant can also beamplified by employing the above-described primer as one primer (in thisregard, however, the product is antisense strands as described above).In addition, the NASBA method in itself is well-known technology, andsince the kit for it is also commercially available, it can be easilycarried out employing the kit in the market.

Other preferable methods for measuring an alternative splicing variantby detecting the presence of exon SV as an indicator include a method inwhich a probe capable of hybridizing with the region comprising thisexon SV under a stringent condition is employed (in situ hybridizationmethod). As for the probe, the one which is capable of hybridizing withthe exon SV region in the nucleic acid having nucleotide sequencerepresented by SEQ ID NO: 1 under a stringent condition, but does nothybridize with the nucleic acid having nucleotide sequence representedby SEQ ID NO: 2 is employed. In order to ensure such specificity,preferable probe is the one in which 20 to 80% of the entire length ofthe probe, more preferably 40 to 60% of the entire length of the probehybridizes with the exon SV region under a stringent condition, and therest portion of the probe hybridizes with adjacent exon 3 region under astringent condition. Size of the probe is not particularly limited,however, in the case of an oligonucleotide, it is usually 10 nucleotidesto the entire length, preferably about 10 nucleotides to about 100nucleotides, and further preferably about 15 nucleotides to about 35nucleotides, and in the case of cRNA or cDNA, it is usually 300nucleotides to 800 nucleotides. In addition, as is the case of primer,it is extremely preferable as a probe to have completely the samenucleotide sequence as the region to where the probe hybridizes,however, 10% or less of nucleotides of full length probe may bereplaced.

The probe may be modified to a labeled probe by binding well-knownlabels, such as fluorescent label, biotin label, radiation label, and anenzyme label to the above-described nucleic acid. In addition, in thecase where the probe is used as a solid-phased probe, the probe may bethe one which has been coupled with any nucleic acid irrelevant to thetest nucleic acid, such as the region to be attached to the solid phase,or a branch in the branched probe for binding with the label.

Probe can be used for a direct measurement of mRNA of alternativesplicing variant contained in the sample to be examined, and also forthe purpose of measuring amplified nucleic acid, after the mRNA isamplified by the nucleic acid amplification method such as the RT-PCR.When amplification product is specifically measured with a probe, toamplify a nucleic acid, primer is set so that exon SV is contained inamplification products. The primer, in this case, is not necessary to bespecific to the alternative splicing variant, but the measurementspecificity is secured by the probe.

The present invention also provides a nucleic acid which is capable ofhybridizing with the nucleic acid comprising the nucleotide sequencerepresented by SEQ ID NO: 1, and even if the nucleic acid comprising thenucleotide sequence represented by SEQ ID NO: 2 is coexisted, does nothybridize with such nucleic acid; or the nucleic acid which, even ifhybridization takes place with the nucleic acid comprising thenucleotide sequence represented by SEQ ID NO: 2, the 3′ terminal thereofbecomes mismatched. Such nucleic acids can be used as a primer and aprobe mentioned above. As such nucleic acids, the nucleic acids whichare capable of hybridizing with the region comprising exon SV in thenucleotide sequence represented by SEQ ID NO: 1, and even if the nucleicacid comprising the nucleotide sequence represented by SEQ ID NO: 2 iscoexisted, does not hybridize, or the nucleic acid which, whenhybridization takes place with the nucleic acid comprising thenucleotide sequence represented by SEQ ID NO: 2, the 3′ terminal thereofbecomes mismatched, are preferable for the specific measurement ofalternative splicing variant. Such nucleic acids of the presentinvention have the number of nucleotide of preferably 10 or more, andparticularly preferably 15 to 35. In addition, the nucleic acids of thepresent invention preferably have the same nucleotide sequence as thenucleotide sequence represented by SEQ ID NO: 1 or partial regionthereof, or the nucleotide sequence in which 10% or less of nucleotidesof the aforementioned nucleotide sequence are replaced, and particularlypreferably is consisted of the same nucleotide sequence as thenucleotide sequence represented by SEQ ID NO: 1 or partial regionthereof.

Such nucleic acid of the present invention can be used as a nucleic acidfor the measurement of alternative splicing variant such as a primer ora probe of the nucleic acid amplification method.

Further, the present invention also provides a measurement kit foralternative splicing variant of OATP1B3 mRNA comprising the abovedescribed nucleic acid for measurement of the present invention. Exceptfor the nucleic acid for measurement of the present invention, thereagents contained in the kit may be known substances, and themeasurement kit can be made up by comprising, for example, in additionto a primer set, reverse transcriptase, Taq DNA polymerase, dNTP, randomprimer, RNase inhibitor, buffer solution, and the like. Alternatively,the kit may be made up of the primer set which consists only of aforward primer and a reverse primer, and as for other reagents, acommercial kit for RT-PCR or the like may be utilized.

The alternative splicing variant mentioned above can be used as a tumormarker. That is, it has been clarified that the alternative splicingvariant provided by the present invention is expressed specifically andstrongly in cancer cell and tissue. Therefore, the measurement methodand the measurement kit provided by the present invention can be appliedfor the detection of cancer. In this regard, type of cancer of detectiontarget is not particularly limited, and can be used, for example, forthe detection of colon cancer, pancreatic cancer, breast cancer, lungcancer, prostatic cancer, esophageal cancer, stomach cancer, livercancer, biliary tract carcinoma, spleen cancer, renal carcinoma, bladdercancer, uterine cancer, ovarian cancer, testicular cancer, thyroidcancer, brain tumor, hematopoietic tumor, and the like. Particularly, asis described in Example mentioned later, it has been confirmed that theOATP1B3/ct is expressed strongly, not only in the human colon cancertissue, also in the cell line derived from human colon cancer (LS180,HCT116) and in the cell line derived from human pancreatic cancer(PK45p). On the other hand, since the expression of OATP1B3/ct was notconfirmed in the tissue of normal human colon origin, the alternativesplicing variant provided by the present invention is particularlyuseful as a marker of colon cancer and pancreatic cancer.

According to another aspect of the present invention, there is provideda method for screening preventive and/or therapeutic agent for cancer.In this screening method, firstly the cancer cell is cultured in thepresence of a test substance. And, for the cultured cell obtained, theabove-described alternative splicing variant of the present invention ismeasured. Subsequently, the obtained measurement results are comparedwith the results of the case where the above-described test substance isabsent and/or evaluated.

Specifically, cancer cell lines derived from various types of carcinomaas described above are cultured in the presence of test substance suchas anticancer drug, candidate anticancer substance, low molecular weightcompound, natural organic polymer substance, extract from naturalanimals and plants, and peptide; and the alternative splicing variant ofthe present invention in the aforementioned cultured cell is measured(detected, measured semi-quantitatively, measured quantitatively); andwhen the form of the alternative splicing variant is decreasedsignificantly compared with the case where the test substance is absent,it is judged that the test substance can be useful as a preventiveand/or therapeutic agent for the cancer from which the above-describedcell line has been derived.

In the screening method of the present invention, screening of apreventive and/or therapeutic agent for human colon cancer can beperformed by using the cell line derived from human colon cancer (forexample, LS180 and HCT116). Similarly, screening of preventive and/ortherapeutic agent for human pancreatic cancer can be performed by usingthe cell line derived from human pancreatic cancer (for example, PK45p).With respect to the cell lines derived from other well-known cancers andtumors, by using the cell lines similarly, screening of preventiveand/or therapeutic agents for various types of cancers and tumors can beperformed.

In addition, in the screening method of the present invention, as forthe method for measuring an alternative splicing variant, theabove-described measurement method and the measurement kit of thepresent invention can be used similarly. Therefore, detailed descriptionis omitted here.

According to the studies carried out by the inventors of the presentinvention, it became clear that at least 4 kinds of polypeptides mightbe expressed from OATP1B3/ct mRNA. Specifically, it was confirmed thatthe polypeptides consisting of respective amino acid sequencesrepresented by SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, and SEQ ID NO:11, respectively, may be expressed from OATP1B3/ct mRNA. Thesepolypeptides are unable to be expressed theoretically from OATP1B3/wtmRNA, and are specific for OATP1B3/ct mRNA. For this reason, thesepolypeptides have usefulness as a tumor marker like OATP1B3/ct mRNA.

From the viewpoint mentioned above, according to another aspect of thepresent invention, there are provided the following polypeptides:

(1) a polypeptide which consists of an amino acid sequence representedby SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11; and

(2) a polypeptide which comprises an amino acid sequence represented bySEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11, andexpression of which is enhanced in cancer cell or cancer tissue, orelse, a polypeptide which comprises an amino acid sequence representedby SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, or SEQ ID NO: 11 in which10% or less of the amino acids are replaced, deleted and/or inserted,and expression of which is enhanced in cancer cell or cancer tissue(also, referred to as “functionally equivalent variant”).

“(2) Functionally equivalent variant” includes “a polypeptide whichcomprises an amino acid sequence represented by SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, or SEQ ID NO: 11, and expression of which is enhancedin cancer cell or cancer tissue”, or “a polypeptide which comprises anamino acid sequence represented by SEQ ID NO: 5, SEQ ID NO: 7, SEQ IDNO: 9, or SEQ ID NO: 11 in which 0 to 10%, preferably 0 to 7%, furthermore preferably 0 to 5%, and particularly preferably 0 to 2% of theamino acids are replaced, deleted and/or inserted, and expression ofwhich is enhanced in cancer cell or cancer tissue”.

“Expression of which is enhanced in cancer cell or cancer tissue” meansthat expression is enhanced by 2 folds or more in cancer cell or cancertissue as compared with the case in non-cancer cell or non-cancertissue.

As mentioned above, although polypeptides of the present aspect wereexplained, hereinafter, the polypeptide which consists of amino acidsequences represented by SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, orSEQ ID NO: 11, and the above-described functionally equivalent variantthereof are also referred to collectively as “polypeptides of thepresent invention”. In addition, among the “polypeptides of the presentinvention”, the polypeptide consisting of amino acid sequencerepresented by SEQ ID NO: 5 is referred to as “f1-1”, the polypeptideconsisting of amino acid sequence represented by SEQ ID NO: 7 isreferred to as “f1-2”, the polypeptide consisting of amino acid sequencerepresented by SEQ ID NO: 9 is referred to as “f2”, the polypeptideconsisting of amino acid sequence represented by SEQ ID NO: 11 isreferred to as “f3”, respectively.

As the polypeptide of the present invention, f1-1 or f3 is particularlypreferable.

According to still another aspect of the present invention, there arealso provided nucleic acids which encode the polypeptides of the presentinvention.

Here, the nucleic acid consisting of nucleotide sequence which encodesthe f1-1 of the polypeptide of the present invention is not particularlylimited, as long as it is the nucleotide sequence which encodes f1-1consisting of amino acid sequence represented by SEQ ID NO: 5 orfunctionally equivalent variant thereof. Preferably, it is the nucleicacid consisting of nucleotide sequence which encodes amino acid sequencerepresented by SEQ ID NO: 5; and further more preferably, it is thenucleic acid consisting of nucleotide sequence represented by SEQ ID NO:4.

Similarly, the nucleic acid consisting of nucleotide sequence whichencodes the f1-2 of the polypeptide of the present invention is notparticularly limited, as long as it is the nucleotide sequence whichencodes f1-2 consisting of amino acid sequence represented by SEQ ID NO:7 or functionally equivalent variant thereof. Preferably, it is thenucleic acid consisting of nucleotide sequence which encodes amino acidsequence represented by SEQ ID NO: 7; and further more preferably, it isthe nucleic acid consisting of nucleotide sequence represented by SEQ IDNO: 6.

In addition, the nucleic acid consisting of nucleotide sequence whichencodes the f2 of the polypeptide of the present invention is notparticularly limited, as long as it is the nucleotide sequence whichencodes f2 consisting of amino acid sequence represented by SEQ ID NO: 9or functionally equivalent variant thereof. Preferably, it is thenucleic acid consisting of a nucleotide sequence which encodes aminoacid sequence represented by SEQ ID NO: 9; and further more preferably,it is the nucleic acid consisting of the nucleotide sequence representedby SEQ ID NO: 8.

In addition, the nucleic acid consisting of nucleotide sequence whichencodes the f3 of the polypeptide of the present invention is notparticularly limited, as long as it is the nucleotide sequence whichencodes f3 consisting of amino acid sequence represented by SEQ ID NO:11 or functionally equivalent variant thereof. Preferably, it is thenucleic acid consisting of nucleotide sequence which encodes amino acidsequence represented by SEQ ID NO: 11; and further more preferably, itis the nucleic acid consisting of nucleotide sequence represented by SEQID NO: 10.

In addition, according to another aspect of the present invention, thereare also provided an expression vector comprising the above-describednucleic acids, a cell which has been transformed by the aforementionedexpression vector.

The expression vector and the cell of the present invention can beproduced by referring to well-known knowledge of prior art. For example,a host cell of eukaryotes or prokaryotes can be transformed byincorporating isolated nucleic acid of the present invention into anappropriate vector DNA once again. In addition, it is possible forrespective host cells to express the nucleic acid by introducing anappropriate promoter and a sequence relevant to the expression ofphenotype into these vectors.

The expression vector of the present invention is not particularlylimited, as long as the nucleic acid of the present invention iscontained, and, for example, the expression vector obtained by insertingthe nucleic acid of the present invention into a known expression vectorselected appropriately according to the host cell to be employed isincluded.

In addition, the cell of the present invention may be, but not limitedparticularly as long as the cell is transfected with the vector of thepresent invention and comprises the nucleic acid of the presentinvention, for example, the cell which has been incorporated with thenucleic acid of the present invention into chromosome of the host cell,or the cell which comprises the nucleic acid of the present invention inthe form of expression vector. Moreover, it may be a cell which isexpressing the polypeptide of the present invention, or may be a cellwhich is not expressing the polypeptide of present invention. The cellof the present invention can be obtained, for example, by transfectingexpression vector of the present invention into a desired host cell.

The desired cell obtained as described above can be cultured accordingto the conventional method, and the polypeptide of the present inventionis produced by the aforementioned culture. As for the medium to beemployed for the aforementioned culture, various kinds of mediumcommonly used depending on the adopted host cell are selectedappropriately.

According to the above description, the polypeptide of the presentinvention produced by the transformed cell can be isolated and purifiedby various kinds of well-known separation procedures through the use ofphysical property, biochemical characteristics, etc. of the polypeptide.

By way of expression after making the marker sequence fused in-frame, itbecomes possible to confirm its expression, to purify it, and so on. Themarker sequence includes, for example, FLAG epitope, Hexa-Histidine tag,Hemagglutinin tag, myc epitope, etc. In addition, by inserting aspecific amino acid sequence which can be recognized by a protease suchas enterokinase, factor Xa, thrombin and the like in between the markersequence and polypeptide, it is also possible to remove the markersequence part by cutting with these enzymes.

The nucleic acid of the present invention, in itself or a part thereofcan be used as a hybridization probe in the method for detecting cancer,and so, these are useful for the detection of cancer. In addition, thepolypeptide of the present invention can be used for the preparation ofspecific antibody for recognizing the polypeptide of the presentinvention, and as a control in the detection and quantitativemeasurement of expression level.

According to the present invention, the antibody which binds to thepolypeptide of the present invention (hereafter, also referred to as“the antibody of the present invention”) can also be provided. Themanufacturing method of the antibody of the present invention is notparticularly limited. The antibody of the present invention (forexample, polyclonal antibody, monoclonal antibody) can be obtained, forexample, by direct administration of the polypeptide which comprises anamino acid sequence represented by SEQ ID NO: 5, and the expression ofwhich is enhanced in cancer cell or cancer tissue, or the polypeptidewhich comprises an amino acid sequence represented by SEQ ID NO: 5 inwhich 1 or several amino acid is replaced, deleted and/or inserted, andthe expression of which is enhanced in cancer cell or cancer tissue, orthe entire of the polypeptide or fragment thereof which consists of theamino acid sequence represented by SEQ ID NO: 5 into various animals.Also, by the method of DNA vaccination using a plasmid which has beenintroduced with the gene encoding the above described polypeptide, theantibody can be obtained (Raz, E. et al., Proc. Natl. Acad. Sci. USA,91, 9519-9523, 1994; Donnelly, J. J. et al., J. Infect. Dis., 173,314-320, 1996).

Polyclonal antibody is produced by emulsifying the aforementionedpolypeptide or fragment thereof in a suitable adjuvant such as Freund'scomplete adjuvant, and administering the emulsion to animal, forexample, rabbit, rat, goat, or chicken, through intraperitoneal,subcutaneous or intravenous route to immunize, then obtaining thepolyclonal antibody from serum or egg of such sensitized animal. Thepolyclonal antibody produced in this way can be isolated and purified bythe conventional protein isolation and purification method. Theconventional protein isolation and purification method includes, forexample, centrifugal separation, dialysis, salting out by ammoniumsulfate, DEAE cellulose, hydroxyapatite, and chromatographic proceduresby protein A agarose and the like.

Monoclonal antibody can be easily produced by the person skilled in theart according to the cell fusion method described by Kohler and Milstein(Kohler, G. and Milstein, C., Nature, 256, 495-497, 1975).

According to still another aspect of the present invention, there isalso provided a nucleic acid which is capable of hybridizing with thenucleic acid comprising the nucleotide sequence represented by SEQ IDNO: 4, SEQ ID NO: 6, SEQ ID NO: 8, or SEQ ID NO: 10 under a stringentcondition, and has at least 15 nucleotides. The “stringent condition” isas mentioned above. Such nucleic acid can be used as a probe fordetecting and for isolating the nucleic acid of the present invention,and also as a primer for amplifying the nucleic acid of the presentinvention. In the case where it is used as a primer, chain length isusually 15 bp to 100 bp, and preferably 15 bp to 40 bp. Preferablenucleotide sequence as a primer includes nucleotide sequence of theprimer used for amplifying each of f1-1, f1-2, f2 and f3 in “9.Preparation of expression plasmid for green fluorescent protein (GFP)fusion ctOATP1B3 peptide” described later in Example. Further, in thecase where it is used as a probe, a DNA which has a part of or wholesequence (or complementary sequence thereof) of the nucleic acid of thepresent invention and a chain length of at least 15 bp is employed.

A probe and a primer employing the above described nucleic acid can beused for detection of cancer.

In addition, based on the present invention, an array of theoligonucleotide probe comprising the nucleotide sequence of the nucleicacid of the present invention or fragment thereof can also beconstructed. Array technique is well-known, and has been used foranalyzing gene expression (Chee, M. et al., (1996) Science, 274,610-613).

The polypeptide of the present invention can also be applied to a methodfor detecting cancer. That is, according to another aspect of thepresent invention, there is provided a method for detecting cancercomprising measuring the amount of the polypeptide of the presentinvention in a sample to be examined which is isolated from livingorganism. On this occasion, it is preferable to employ theaforementioned antibody of the present invention. In addition, adetection kit for cancer comprising the aforementioned antibody can alsobe provided. Hereinafter, this embodiment will be explained in moredetail.

Detection of the polypeptide of the present invention in a sample to beexamined can be performed, for example, by subjecting the sample to beexamined to a method constructed by combining various molecular weightmeasurement methods such as gel electrophoresis, various kinds ofseparation and purification methods (e.g. ion exchange chromatography,hydrophobic chromatography, affinity chromatography, reverse phasechromatography, and the like), ionization methods (e.g. electron impactionization method, field desorption method, secondary ionization method,fast atom bombardment method, matrix assisted laserdesorption/ionization (MALDI) method, electrospray ionization method,and the like), and mass spectrometers (e.g. double-focusing massspectrometer, quadrupole mass spectrometer, time-of-flight massspectrometer, Fourier transform mass spectrometer, ion-cyclotron massspectrometer, and the like), and by detecting a band or a spot, or apeak corresponding to the molecular weight of the polypeptide of thepresent invention, but is not limited thereto. Since the amino acidsequence of the polypeptide of the present invention is known, a methodfor detecting the polypeptide of the present invention by preparing anantibody which recognizes the aforementioned amino acid sequence, andcarrying out western blotting or various immunoassays using the antibodycan be employed more preferably. In addition, the method for preparingthe aforementioned antibody is as mentioned above. The detection methodfor the polypeptide of the present invention using the antibody of thepresent invention is particularly useful in that the polypeptide of thepresent invention can be detected with high sensitivity and precisenesswithout employing special equipment like mass spectrometer, if anoptimized immunoassay system is developed and constructed as a kit.

The detection method for cancer of the present invention using theantibody of the present invention is not particularly limited, and anymeasurement method can be employed, as long as it is a measurementmethod in which the amount of antibody, antigen or antibody-antigencomplex corresponding to the amount of antigen in the sample to beexamined is detected by chemical or physical procedures, and computedfrom a standard curve which is made using standard solutions containingknown amounts of the antigen. For example, nephelometry, competitionmethod, immunometric method, sandwiching method, and the like are usedsuitably.

As to a labeling agent to be used in the measurement method using alabeling substance, for example, radioisotope, enzyme, fluorescentsubstance, luminescent substance, and the like are employed. As toradioisotope, for example, [125I], [131I], [3H], [14C], and the like areused. As to the above-described enzyme, the one which is stable and hasa high specific activity is preferable, and, for example,β-galactosidase, β-glucosidase, alkaline phosphatase, peroxidase, malatedehydrogenase, and the like are used. As to fluorescent substance, forexample, fluorescamine, fluorescein isothiocyanate, and the like areused. As to luminescent substance, for example, luminol, luminolderivatives, luciferin, lucigenin, and the like are used. Furthermore,biotin-avidin system can also be employed for binding of antibody orantigen and labeling agent.

For the purpose of insolubilization of antigen or antibody, physicaladsorption may be used, or a method using chemical bond which is usuallyused for insolubilizing or immobilizing protein or enzyme may be used.The carrier includes insoluble polysaccharides such as agarose, dextran,and cellulose, synthetic resin such as polystyrene, polyacrylamide, andsilicone, or glass, and so on.

In the sandwich technique, the insolubilized antibody of the presentinvention is reacted with the sample to be examined (primary reaction),and further reacted with labeled another antibody of the presentinvention (secondary reaction), after that, an amount (activity) of thelabeling agent on the insolubilized carrier is measured, and thereby anamount of the peptide of the present invention in the sample to beexamined can be measured quantitatively. The primary reaction and thesecondary reaction may be carried out in reverse order, or may beperformed simultaneously or with a certain interval.

The monoclonal antibody against the polypeptide of the present inventioncan also be used for measurement system other than the sandwichtechnique, for example, the competition method, the immunometricmethods, or the nephelometry, and so on.

In the competition method, after reacting the antigen in a sample to beexamined and the labeled antigen competitively with the antibody,unreacted labeled antigen (F) and labeled antigen (B) bound with theantibody are separated (B/F separation), and the amount of label ofeither B or F is measured, to determine the amount of the antigen in thesample to be examined. For this reaction method, liquid phase method inwhich a soluble antibody is used as the antibody, and B/F separation iscarried out using polyethylene glycol and a secondary antibody againstthe aforementioned antibody, and the like, and solid phase method inwhich a solid-phased antibody is used as a primary antibody, or asoluble antibody is used as a primary antibody and a solid-phasedantibody is used as the secondary antibody, are employed.

In the immunometric method, after the antigen in the sample to beexamined and the solid-phased antigen are reacted competitively with acertain amount of labeled antibody, the solid phase and the liquid phaseare isolated, or else, the antigen in the sample to be examined isreacted with an excess amount of labeled antibody, then the solid-phasedantigen is added to bind unreacted labeled antibody to the solid phase,after that the solid phase and the liquid phase are separated.Subsequently, an amount of label of any phase is measured, to determinethe amount of antigen in the sample to be examined quantitatively.

In addition, in the nephelometry, the amount of the insoluble sedimentproduced within gel or in solution as a result of the antigen-antibodyreaction is measured. Even when the amount of antigen in the sample tobe examined is small, and only a small amount of sediment is obtained, alaser nephelometry using dispersion of laser and the like is employedsuitably.

In applying these respective immunological measurement methods to thedetection method of the present invention, any setup of particularconditions, operations, and the like is not required. The measurementsystem for the polypeptide of the present invention may be constructedby adding the usual technical considerations of those skilled in the artto the usual conditions and operations in the respective methods. As tothe details of these general technical procedures, review articles,books, etc. can be referred to. For example, Hiroshi Irie ed.,“Radioimmunoassay” (Kodansha, published in Showa 49); Hiroshi Irie ed.,“Continued Radioimmunoassay” (Kodansha, published in Showa 54); EijiIshikawa, et al. ed., “Enzyme-Linked Immunosorbent Assay” (Igaku-Shoin,published in Showa 53); Eiji Ishikawa, et al. ed., “Enzyme-LinkedImmunosorbent Assay”, (2nd Edition) (Igaku-Shoin, published in Showa57); Eiji Ishikawa, et al. ed., “Enzyme-Linked Immunosorbent Assay”,(3rd Edition) (Igaku-Shoin, published in Showa 62); “Methods inENZYMOLOGY” Vol. 70 (Immunochemical Techniques (Part A)), ibid. Vol. 73(Immunochemical Techniques (Part B)), ibid. Vol. 74 (ImmunochemicalTechniques (Part C)), ibid. Vol. 84 (Immunochemical Techniques (Part D:Selected Immunoassays)), ibid. Vol. 92 (Immunochemical Techniques (Part:Monoclonal Antibodies and General Immunoassay Methods)), ibid. Vol. 121(Immunochemical Techniques (Part I: Hybridoma Technology and MonoclonalAntibodies)) (the foregoing are published from Academic Press), etc. canbe referred to.

Alternatively, another detection method for the present invention usingthe antibody of the present invention includes a method in which theaforementioned antibody is fixed on the surface of the probe which canconform to the aforementioned mass spectrometer; a sample to be examinedis allowed to contact with the aforementioned antibody on theaforementioned probe; and the biological sample component caught by theaforementioned antibody is subjected to mass spectrometry; to detect apeak corresponding to the molecular weight of the marker peptide whichis recognized by the aforementioned antibody.

When the polypeptide of the present invention is detected in a sample tobe examined with statistically significant difference from the level inthe control sample by any one of the above described methods, it can bediagnosed that possibility of comprising cancer cell or cancer tissue inthe sample to be examined is high.

According to still another aspect of the present invention, a medicamentis provided. The aforementioned medicament comprises the above-describedpolypeptide of the present invention or fragment thereof, or the nucleicacid of the present invention or fragment thereof, which is to be usedfor inducing specific cytotoxic T-cell (CTL). The aforementionedmedicament is preferably cancer vaccine. Hereafter, this embodiment willbe explained in more detail.

When the medicament of the present invention comprises the polypeptideof the present invention or fragment thereof, it is necessary that theaforementioned polypeptide or fragment thereof can induce specificcytotoxic T-cell (CTL) by binding with HLA. From this point of view, itis preferable that the aforementioned polypeptide or fragment thereofconsists of consecutive 8 to 11 amino acid residues in the polypeptideof the present invention. It should be noted that the polypeptidefragment inducing specific CTL can easily be prepared by referring tothe well-known knowledge in the art.

“Can induce specific cytotoxic T-cell (CTL) by binding with HLA” meansthat the polypeptide of the present invention or fragment thereof, orthe nucleic acid of the present invention or fragment thereof forms acomplex by binding with HLA, and such complex can be recognized by theCTL. In other words, it means that the polypeptide of the presentinvention or fragment thereof, or the nucleic acid of the presentinvention or fragment thereof has binding activity with HLA, and has anactivity to induce a specific CTL in the form of a complex with HLA.

On the other hand, when the medicament of the present inventioncomprises the nucleic acid of the present invention or fragment thereof,the polypeptide which has an amino acid sequence encoded by theaforementioned nucleic acid is recognized in itself by CTL, and canactivate the aforementioned CTL, or can provide a polypeptide fragmenthaving such activity, and can function as a tumor antigen. As for thenucleic acid or fragment thereof employed in the present aspect, it ispreferable that they are the polynucleotide or complementary strandthereof which consists of at least 24 or more of nucleotidescorresponding to the region coding the polypeptide of the presentinvention. Such a polynucleotide can be selected by checking theexpressed peptides, for example, using known protein expression system.

The medicament of the present invention can be used, for example, as acancer vaccine. Although only one kind of polypeptide or fragmentthereof, or nucleic acid or fragment thereof is effective as a cancervaccine, it is preferable that plural kinds of them are used incombination. This is because it is expected to be more effective to useplural types of tumor antigens in combination as a vaccine, because CTLof cancer patients is a cell population which recognizes severaldifferent kinds of tumor antigens. In addition, the polypeptide of thepresent invention and the nucleic acid of the present invention may beused in conjunction with a polypeptide or a nucleic acid other thanthese.

The cancer vaccine provided by the present invention can be used in thepresence or absence of an appropriate adjuvant, independently or bybinding with pharmaceutically acceptable carrier. The carrier is notparticularly limited as long as it does not give any hazardous effect onhuman body, and, for example, cellulose, polymerized amino acid,albumin, and the like can be used. As to dosage form, the dosage formwhich is well known for peptide formulation can be selected. As todosage, it may vary depending on the recognition property of CTL,disorder to be treated, age of patient, body weight and so on, however,in the case of peptide, it is usually 0.0001 mg to 1000 mg/day/adulthuman, preferably 0.001 mg to 1000 mg/day/adult human, more preferably0.1 mg to 100 mg/day/adult human, further more preferably 0.1 mg to 10mg/day/adult human as an active entity. This medication may beadministered once a day, once a week, or once several months.

The medicament of the present invention can also be utilized forincorporating the polypeptide of the present invention or fragmentthereof into an appropriate vector, and for introducing the vector invivo or ex vivo. The vector includes, for example, retrovirus,adenovirus, vaccinia virus, and the like, and retroviral system ispreferable. Dosage may vary depending on the recognition property ofCTL, however, it is 0.1 μg to 100 mg/day/adult human, and preferably 1μg to 50 mg/day/adult human, as a DNA content. This medication may beadministered once a day, once a week, or once several months.

EXAMPLE

Hereinafter, the present invention will be explained more specificallyby means of Examples and Reference Examples, however, technical scope ofthe present invention shall not be limited thereto.

1. Primer

The primers used for genomic DNA contamination check, RNAligase-mediated rapid amplification of 5′ cDNA end (RLM-RACE), reversetranscription PCR (RT-PCR), cDNA cloning, quantitative real-time PCR,and the primers used for peptide expression from the GFP fusion peptideexpression plasmid were synthesized by Invitrogen (Carlsbad, Calif.,USA) or Greiner-Bio-One (Taufkirchen, Germany) on consignment contract,and each of them has nucleotide sequence shown in Tables 1 to 6 below.

TABLE 1 Primer Used for Genomic DNA Contamination Check PrimerNucleotide sequence (5′ 3′) GAPDH F TGCACCACCAACTGCTTA (SEQ ID NO: 12)GAPDH R GGATGCAGGGATGATGTTC (SEQ ID NO: 13) F: forward; R: reverse

TABLE 2 Primer Used for RLM-5′-RACE Primer Nucleotide sequence (5′ 3′)SLCO1B3 648R GCCACGAAGCATATTCCCCATGAAG (SEQ ID NO: 14) SLCO1B3 424RnestTTCCAGTTCCCATAAGGAGACAACC (SEQ ID NO: 15) R: reverse

TABLE 3 Primer Used for RT-PCR Annealing temperature PrimerNucleotide sequence (5′ 3′) (° C.) SLCO1B3 TSS in GTGCAAGTCACAGGGGATGGGA 60 cancer 33F (SEQ ID NO: 16) SLCO1B3 TSS in GCTGAATGACAGGGCTGCCAAG 60 cancer 145R (SEQ ID NO: 17) F: forward; R:reverse

TABLE 4 Primer Used for cDNA Cloning of OATP1B3/ct and 5 OATP1B3/wtPrimer Nucleotide sequence (5′ 3′) ctSLCO1B3 cloningGTGCAAGTCACAGGGGATGGGA F33 (SEQ ID NO: 16) ctSLCO1B3 cloningGCAATGTTAGTTGGCAGCAGCA 2149R (SEQ ID NO: 18) SLCO1B3 F27GGTATCTGTAGTTTAATAATGGACC (SEQ ID NO: 19) SLCO1B3 R2184GAAAGACCAGGAACACCTCA (SEQ ID NO: 20)

TABLE 5 Primer Used for Quantitative Real-Time PCR PrimerNucleotide sequence (5′ 3′) ctSLCO1B3 Left 59-76 TTGGCTTGGGCTCAGAGA(SEQ ID NO: 21) ctSLCO1B3 Right 130-151 TGCCAAGAACATCTGCTAGTTT(SEQ ID NO: 22) SLCO1B3 F153 for AACAGCAGAGTCAGCATCTTCAG real-time(SEQ ID NO: 23) SLCO1B3 R194 for AACATCTTGAATCCATTGCAGC real-time(SEQ ID NO: 24) F: forward; R: reverse

TABLE 6 Primer Used for Peptide Expression from the GFP Fusion Peptide Expression Plasmid Primer Nucleotide sequence (5′ 3′) Frame 1-1ct1B3 f1 45-78+GFP CCtctagaGGGATGGGATGGCTTGGCTTGGGCTCAGAGACCGTGAGCAAGGGCG(SEQ ID NO: 25) GFP L696+KpnIGGggtaccTTACTTGTACAGCTCATCCATGCCG (SEQ ID NO: 26) Frame 1-2ctOATP1B3 cloning GCtctagaGTGCAAGTCACAGGGGATGGGA U33+XbaI(SEQ ID NO: 27) ct1B3 f1 L153+BamHI GTggatccGCAATATAGCTGAATGACAGGG(SEQ ID NO: 28) Frame 2 ctOATP1B3 cloning GCtctagaGTGCAAGTCACAGGGGATGGGAU33+XbaI (SEQ ID NO: 27) ct1B3 f2 L239+BamHI TAggatccACCAGCAAGAGAAGAGGA(SEQ ID NO: 29) Frame 3 ctOATP1B3 cloning GCtctagaGTGCAAGTCACAGGGGATGGGAU33+XbaI (SEQ ID NO: 27) ct1B3 f3 L160+BamHITCggatccGTGCTTTAGCAATATAGCTGAAT (SEQ ID NO: 30) GFP   GFP U4+BamHICGggatccGTGAGCAAGGGCGCCGAGC (SEQ ID NO: 31) GFP L696+KpnIGGggtaccTTACTTGTACAGCTCATCCATGCCG (SEQ ID NO: 32) *Lower-cases indicaterecognition sites of restriction enzymes.2. Cell Culture Method

LS180 cell which is a cell line derived from human colon cancer waspurchased from Dainippon Sumitomo Pharma Co. Ltd. (Osaka, Japan). HCT116cell which is a cell line derived from human colorectal cancer waskindly bestowed from Dr. Vogelstein (Johns Hopkins University,Baltimore, Md., USA). PK45p cell which is a cell line derived from humanpancreatic cancer was obtained from Cell Resource Center for BiomedicalResearch, Institute of Development, Aging and Cancer, Tohoku University(Sendai, Japan). LS180 cell and PK45p cell were each cultured in MinimumEssential Medium (MEM) (Invitrogen) or RPMI (Roswell Park MemorialInstitute) 1640 medium (Invitrogen) supplemented with 10%heat-inactivated fetal bovine serum (FBS) (Gemini Bio-Products, CA,USA), and 50 U/mL penicillin-50 μg/mL streptomycin (Invitrogen), in anatmosphere of 5% CO₂/95% air, at 37° C. in a CO₂ incubator.

3. Experimental Sample

Caucasian liver sample was obtained from the HAB (Human and AnimalBridging) Research Organization (Tokyo, Japan). The above-describedsample is transplantation-incompatible human liver tissue which wasimported from the U.S. through National Disease Research Interchange(NDRI) (Philadelphia, Pa., USA). In addition, the above-described livertissue used was not suffered from infectious diseases such as hepatitis.In addition, the research use of these specimens in the present studieswas approved in advance by the Ethics Committee, Graduate School ofPharmaceutical Sciences, Chiba University.

4. Extraction of Total RNA

Extraction of total RNA from cell was performed using FastPure™ RNAIsolation Kit (Takara Bio Inc., Shiga, Japan) according to the protocol.No contamination of genomic DNA in the extracted total RNA was confirmedby the PCR employing 20 μL of reaction solution which contains 10 μL ofGoTaq Green Master Mix (Promega Corporation, Madison, Wyo., USA) andeach 0.5 μl of primers (human glyceraldehyde-3-phosphate dehydrogenase(GAPDH) F and GAPDH R, as shown in the above Table 1), and 1 μL of RNA.The PCR was carried out as follows: after heating the reaction solutionat 95° C. for 30 seconds, a reaction cycle composed of heating at 95° C.for 10 seconds, at 50° C. for 10 seconds and at 72° C. for 30 secondswas repeated for 40 times. Also, the extraction of the total RNA fromthe Caucasian liver was carried out according to the protocol usingFastPure™ RNA Isolation Kit. A pair of cancerous/normal colon total RNAwas purchased from Applied Biosystems Inc. (Foster city, MA, USA).

5. Determination of Transcription Starting Point of SLCO1B3 Gene

Transcription starting point of SLCO1B3 gene was determined by theRLM-5′-RACE method using GeneRacer™ Kit (Invitrogen). The determinationwas performed using the total RNA derived from human colorectal canceror the total RNA derived from Caucasian liver as a template according tothe protocol. For the PCR reaction, a 20 μL of PCR reaction solutioncontaining 10 μL of GoTaq Green Master Mix, 1 μL of reversetranscriptase template, and each 0.5 μL of 10 μM GeneRacer™ 5′ primerand reverse gene specific primer (“SLCO1B3 648R”, as shown in the aboveTable 2) was prepared. After heating the reaction solution at 94° C. for2 minutes, Touch-down PCR in which a reaction cycle composed of heatingat 94° C. for 30 seconds, at 68° C. for 30 seconds and at 72° C. for 1minute was repeated 5 times; a reaction cycle composed of heating at 94°C. for 30 seconds, at 66° C. for 30 seconds and at 72° C. for 1 minutewas repeated 5 times; a reaction cycle composed of heating at 94° C. for30 seconds, at 65° C. for 30 seconds and at 72° C. for 1 minute wasrepeated 30 times were carried out consecutively, was performed. Afterthis Touch-down PCR, Nested-PCR was performed using the PCR reactionsolution diluted 5 times with sterile water as a template. For theNested-PCR reaction, 20 μL of PCR reaction solution containing 10 μL ofGoTaq Green Master Mix, 1 μL of template, and each 0.5 μL of 10 μMGeneRacer™ 5′Nested primer and reverse Nested gene specific primer(“SLCO1B3 424Rnest”, as shown in the above Table 2) was prepared. Afterheating this reaction solution at 95° C. for 30 seconds, a reactioncycle composed of heating at 95° C. for 10 seconds, at 53° C. for 10seconds and at 72° C. for 30 seconds was repeated for 40 times. The PCRproducts was subjected to 3% agarose gel electrophoresis, and visualizedby ethidium bromide staining. Results of the electrophoresis are shownin FIG. 3. As shown in FIG. 3, with the sample obtained by using totalRNA derived from Caucasian liver as a template, a band corresponding tothe reported transcription starting point (“TSS” as shown in FIG. 1) wasconfirmed. On the other hand, with the sample obtained by using totalRNA derived from human colorectal cancer as a template, a band with anucleotide length shorter than that derived from the reportedtranscription starting point was identified. From these results, it wassuggested that, in the OATP1B3 mRNA derived from human colorectalcancer, different transcription starting point from that alreadyreported (locating down-stream (3′) side from the reported point)exists. In addition, “Non-template control” shown in FIG. 3 is anegative control which does not include template (“NTC” in FIG. 5mentioned later is the same meaning).

Subsequently, after separation and purification was carried out, the DNAfragment comprising the Nested-PCR product obtained above was insertedin pTAC-1 vector using DynaExpress TA PCR Cloning Kit (BioDynamicsLaboratory Inc., Tokyo, Japan), and selected using ampicillin. PlasmidDNA was refined using Plasmid Miniprep Kit (Bio-Rad Laboratories Inc.).The nucleotide sequence of the obtained plasmid DNA was subjected to acycle-sequencing reaction using CEQ™ DTCS-Quick Start Mix (BeckmanCoulter, Inc., Fullerton, Calif., USA), and analyzed using CEQ 2000 XLDNA analysis system (Beckman Coulter, Inc.), to identify the newtranscription starting point of SLCO1B3 gene.

When the nucleotide sequence near the new transcription starting pointof SLCO1B3 gene was compared with the database of human genomic DNA, itbecame evident that a novel transcription starting point exists in theregion of the intron 2 of SLCO1B3 gene, and further a new exon (exon SV)exists in this region. Moreover, this exon SV has been spliced to theexon 3 of OATP1B3/wt (see FIG. 4).

6. Preparation of cDNA, and Analysis of mRNA Expression by RT-PCR Method

The total RNA extracted from LS180 cell, PK45p cell by the proceduredescribed in the above Item 4, a pair of cancerous/normal colon totalRNA and the total RNA derived from the Caucasian liver were used as atemplate; the reverse transcriptase reaction was carried out using HighCapacity cDNA Reverse transcriptase kit (Applied Biosystems Inc.); andrespective cDNAs were prepared. The RT-PCR was carried out using thiscDNA as a template, and the expression level of new OATP1B3 mRNA wasanalyzed. As for the PCR, 20 μL of PCR reaction solution containing 10μL of GoTaq Green Master Mix, 1 μL of template cDNA, and each 0.5 μL of10 μM forward primer and reverse primer (“SLCO1B3 TSS in cancer 33F” and“SLCO1B3 TSS in cancer 145R”, as shown in the above Table 3) wasprepared. After heating this reaction solution at 95° C. for 30 seconds,a reaction cycle composed of heating at 95° C. for 10 seconds, atannealing temperature suitable for respective primers (“annealingtemperature”, as shown in the above Table 3) for 10 seconds and at 72°C. for 15 to 20 seconds was repeated for 38 times. The PCR product wassubjected to 3% agarose gel electrophoresis, and visualized by ethidiumbromide staining. Results of the electrophoresis are shown in FIG. 5. Asshown in FIG. 5, in addition to human colon cancer tissue, expression ofnovel OATP1B3 mRNA was confirmed in LS180 cell and HCT116 cell eachderived from colon cancer and PK45p cell derived from pancreatic cancer,as well. On the other hand, expression of this mRNA was not identifiedin the normal human colon tissue. From these results, since this novelOATP1B3 mRNA was considered to be a molecular species expressed highlyin cancer cell/tissue, it was named as “OATP1B3/ct”.

7. cDNA Cloning of OATP1B3/ct

The cDNA cloning of OATP1B3/ct was carried out by the PCR using cDNAderived from human colorectal cancer as a template, and using a primerset (ctSLCO1B3 cloning F33 and ctSLCO1B3 cloning 2149R) shown in theabove Table 4 and PrimeSTAR HS DNA polymerase (Takara Bio Inc., Shiga,Japan). In addition, as shown in Table 4, the forward primer employedfor the cDNA cloning of OATP1B3/ct was identical to that of “SLCO1B3 TSSin cancer 33F” employed for the RT-PCR of the above-described “6.Preparation of cDNA, and analysis of mRNA expression by RT-PCR method”.The PCR was carried out under the conditions that after heating at 94°C. for 3 minutes, 40 cycles of sequential heating at 98° C. for 10minutes, 58° C. for 5 seconds, and 72° C. for 2 minutes and 30 seconds,and 72° C. for 3 minutes as the final step were repeated. The PCRproduct obtained was isolated by 1% agarose gel electrophoresis andrefined by extraction, then inserted into pCR Blunt-II TOPO vector(Invitrogen Corporation). By processing the obtained construct by XbaI(Nippon Gene Co., Ltd., Tokyo, Japan) and BamHI (Nippon Gene Co., Ltd.),OATP1B3/ct cDNA was clipped, and the obtained cDNA was allowed toligation with the similarly processed pcDNA3.1(−) Neo vector (InvitrogenCorporation), to produce OATP1B3/ct/pcDNA3.1(−).

In addition, the cDNA cloning of OATP1B3/wt was also performed by thesame procedures. Specifically, the cloning was carried out by the PCRusing cDNA derived from Caucasian liver as a template, and using aprimer set (SLCO1B3 F27 and SLCO1B3 cloning 218R) shown in the aboveTable 4 and KOD-plus-Polymerase (TOYOBO Co., LTD., Osaka, Japan). ThePCR reaction was carried out under the conditions that after heating at94° C. for 3 minutes, 35 cycles of sequential heating at 94° C. for 30minutes, 48° C. for 30 seconds, and 68° C. for 2 minutes and 30 seconds,and 68° C. for 5 minutes as the final step were repeated. The PCRproduct obtained was isolated by 1% agarose gel electrophoresis andrefined by extraction, then inserted into pCR Blunt-II TOPO vector(Invitrogen Corporation). By processing the obtained construct by ApaI(Nippon Gene Co., Ltd.) and BamHI, OATP1B3/wt cDNA was clipped, and theobtained cDNA was allowed to ligation with the similarly processedpcDNA3.1(−) Neo vector, to produce OATP1B3/wt/pcDNA3.1(−).

The nucleotide sequences of OATP1B3/ct and OATP1B3/wt in two plasmidsprepared as described above were analyzed using CEQ 2000 Dye terminatorCycle Sequencing with Quick Start Kit (Beckman Coulter, Inc.) and CEQ2000 XL DNA analysis system (Beckman Coulter, Inc.). In FIG. 1, cDNAsequence of OATP1B3/ct (SEQ ID NO: 1) measured herewith is showntogether with the cDNA sequence of OATP1B3/wt (Refseq Accession No.NM_019844, SEQ ID NO: 2). In addition, as shown in FIGS. 1 and 4, it canbe confirmed that, in OATP1B3/ct, the exon 1 and exon 2 of OATP1B3/wtare deleted, and instead, exon SV exists and splicing to the exon 3 ofOATP1B3/wt.

8. Analysis of mRNA Expression by Quantitative Real-Time PCR

Using GHL pool (cDNA pool prepared from five Japanese liver samples)which was prepared by the procedure described in the above item 6, HHLpool (cDNA pool prepared from five Caucasian live samples), cDNA derivedfrom each human colorectal cancer tissue, LS180 cell, K45p cell, andHCT116 cell, and using plasmid prepared according to the proceduredescribed in the above item 7, quantitative real-time PCR was performed,and quantitative measurement of each mRNA of OATP1B3/ct and OATP1B3/wtwas carried out using standard curve method. For the detection of PCRand DNA amplification, ABI PRISM® 7000 (Applied Biosystems Inc.) wasemployed.

For the detection of OATP1B3/ct mRNA, a 20 μL of PCR reaction solutioncontaining 1 μL of template cDNA, 10 μL of 2× FastStart Universal ProbeMaster (ROX) (Roche Diagnostics, GmbH, Penzberg, Germany), 0.2 μL ofUniversal Probe #59 (Roche Diagnostics, GmbH), and each 0.4 μL of aprimer set (ctSLCO1B3 Left 59-76, and ctSLCO1B3 Right 130-151) shown inthe above Table 5 was prepared. The detection was performed by 2-stepPCR in which, after heating the reaction solution at 95° C. for 5minutes, the cycle of heating at 95° C. for 15 seconds and at 60° C. for1 minute was repeated for 45 times.

On the other hand, for the detection of OATP1B3/wt mRNA, a 20 μL of PCRreaction solution containing 1 μL of template cDNA, 10 μl of 2×SYBERPremix Ex Taq II (Takara Bio Inc.), 0.4 μL of 50×ROX Reference Dye(Takara Bio Inc.), and each 0.8 μL of a primer set (SLCO1B3 F153 forreal-time, and SLCO1B3 R194 for real-time) shown in the above Table 5was prepared. The detection was performed by 2-step PCR in which, afterheating the reaction solution at 95° C. for 20 seconds, the cycle ofheating at 95° C. for 5 seconds and at 60° C. for 33 seconds wasrepeated for 40 times.

As to the sample for standard curve, the sample for the measurement ofOATP1B3/ct mRNA was prepared by using OATP1B3/pTOPO prepared by theprocedure described in the above item 7. as a template, and using 5points in a range of 2.86×10⁻²³ to 1.43×10⁻¹⁷ mole per well; on theother hand, the sample for the measurement of OATP1B3/wt mRNA wasprepared by using OATP1B3/pcDNA3.1(−) prepared by the proceduredescribed in the above item 7. as a template, and using 5 points in arange of 1.09×10⁻²³ to 1.09×10⁻¹⁷ mole per well. Detection of PCR andDNA amplification was performed as in the case with the cDNA samples.Moreover, the standard curve was obtained by linear regression based onthe least-squares method.

The mRNA expression profiles obtained as a result of the real timequantitative PCR is shown in FIG. 6. FIG. 6-A shows expression levels ofOATP1B3/ct in respective tissues and cells. Also, FIG. 6-B showsexpression levels of OATP1B3/wt in respective tissues and cells. Inaddition, the bar shown in FIG. 6 represents number of copies of OATP1B3mRNA as average value±standard deviation in three independentexperiments. Furthermore, FIG. 6-C shows ratio of expression level (copynumber of OATP1B3/ct versus copy number of OATP1B3/wt).

As shown in FIG. 6-A, copy number of OATP1B3/ct mRNA present in 1 ng oftotal RNA was 8 for the GHL pool (Japanese liver), and it was 4 for theHHL pool (Caucasian liver). In contrast, in human colorectal cancertissue, h colon Tu #1 and #2, it was 1812 and 89, respectively, and 2411for LS180 cell, 289 for PK45p cell, and 644 for HCT116 cell. On theother hand, copy number of OATP1B3/wt mRNA was 12187 for GHL pool and7473 for HHL pool, and in human colorectal cancer tissue, h colon Tu #1and #2, it was 30 and 3, respectively, and 865 for LS180 cell, 2 forPK45p cell, and 172 for HCT116 cell. Therefore, the amount of OATP1B3/ctmRNA expression was, in comparison with the amount of OATP1B3/wt mRNAexpression, 60 times and 30 times higher in human colorectal cancertissue h colon Tu #1 and #2, respectively, 2.8 times higher in LS180cell, 144.5 times higher in PK45p cell, and 3.7 times higher in HCT116cell (FIG. 6-C). On the other hand, in GHL pool and HHL pool, the amountof expression of OATP1B3/ct mRNA was about 1/1500 and about 1/1700 ofthe amount of expression of OATP1B3/wt mRNA, respectively. From theresults obtained above, it was shown that OATP1B3/ct mRNA was almostspecifically expressed in human colorectal cancer tissue, and inaddition, in the cells derived from human colorectal cancer alsoexpressed OATP1B3/ct mRNA very dominantly. That is, by the inventors ofthe present application, it was clarified that what was expressed incancer cell and tissue was not the previously reported OATP1B3/wt, butan alternative splicing variant which was newly discovered this time.

9. Preparation of Expression Plasmid for Green Fluorescent Protein (GFP)Fusion ctOATP1B3 Peptide

As shown in FIG. 7, a putative open reading frame (ORF) is present ineach frame of OATP1B3/ct mRNA. It is presumed that two ORFs of frame 1(f1-1 and f1-2) encode a peptide which consists of 10 amino acids and apeptide which consists of 27 amino acids, respectively. In addition, itwas presumed that ORF of frame 2 encodes a protein consisting of 655amino acids, which uses the midway of the frame of OATP1B3/wt as aninitiation codon and share as it is as a stop codon of OATP1B3/wt (f2).Further, it was presumed that the ORF of frame 3 encodes the peptidewhich consists of 43 amino acids (f3). To analyze expression of thesetranslation product, the plasmid which expresses GFP-fused protein(f1-1/GFP, f1-2/GFP, f2/GFP, f3/GFP) on each C-terminal side of thepeptide coding region (f1-1, f1-2, f3) which exists in the frame 1 andframe 3 of OATP1B3/ct mRNA, and on the C-terminal side of N-terminal 20amino acid region of the protein coding region (f2) was prepared.

Using OATP1B3/ct/pcDNA3.1(−) as a template, and using a primer shown inTable 6, F1-2 region, f2 region, and f3 region were amplified by PCR(after 95° C. for 30 seconds, a cycle of 95° C. for 10 seconds, 60° C.for 10 seconds, and 72° C. for 20 seconds was repeated 40 times, and 72°C. for 10 seconds was the final step). In addition, as to GFP andf1-1/GFP, using primer shown in Table 6, and using pAcGFP-c1 as atemplate, amplification was carried out by PCR (after heating at 94° C.for 3 minutes, a cycle of 98° C. for 10 seconds, 59° C. for 5 seconds,and 72° C. for 50 seconds was repeated 40 times, and 72° C. for 3minutes was the final step). The PCR product of GFP was processed withBamHI, and then ligation was carried out with each similarlyBamHI-processed PCR product of f1-2, f2 and f3. Using them as atemplate, and using the primer shown in Table 6, amplification wasperformed by the PCR (after heating at 94° C. for 2 minutes, a cycle of94° C. for 10 seconds, 60° C. for 30 seconds, and 72° C. for 60 secondswas repeated 35 times, and 72° C. for 30 seconds was the final step).Each of f1-2/GFP and f3/GFP obtained was inserted in pTAC-1 vector usingDynaExpress TA PCR Cloning Kit (BioDynamics Laboratory Inc., Tokyo,Japan). Also, each of f1-1/GFP and f2/GFP obtained was inserted in pCRBlunt-II TOPO vector (Invitrogen Corporation). These plasmids wereprocessed with XbaI and KpnI, and then ligation was carried out withsimilarly processed pcDNA3.1(−)Neo, to produce f1-1/GFP/pcDNA3.1,f1-2/GFP/pcDNA3.1, f2/GFP/pcDNA3.1, and f3/GFP/pcDNA3.1.

10. Reverse Transfection

To a mixture of 320 ng/well of mCherry/pcDNA3.1(−) and 320 ng/well ofeach GFP fusion peptide expression plasmid or empty pcDNA3.1(−)Neo(mock), 100 μL/well of OPTI-MEM®(Invitrogen Corporation) and 0.5 μL/wellof Plus Reagent (Invitrogen Corporation) were mixed, and 1.6 μL/well ofLipofectamin LTX (Invitrogen Corporation) was added thereto, andtogether with this, 3.5×10⁵ cell/well of 293FT cell was seeded to 24well plate. The green fluorescence of the GFP origin and the redfluorescence of the mCherry origin were observed after 24 hours fromseeding using Olympus Fluoview ver2.3 (Olympus Corporation, Tokyo,Japan). Results are shown in FIG. 8. Here, in FIG. 8, green color is thefluorescence of the GFP origin and red color is the fluorescence of themCherry origin which is transfection control. Moreover, superposition ofboth fluorescences is shown as Merge and a differential interferenceimage is shown as Phase. In addition, all laser intensity and exposuretime employed for analysis were made equal.

As shown in FIG. 8, the strong fluorescence was observed in thetransfected 293FT cell which is considered to be f1-1/GFP and f3/GFPorigin (FIGS. 8 (b) and (e)). In addition, the fluorescence of f2/GFPorigin was also observed (FIG. 8 (d)). On the other hand, very weakfluorescence was observed from f1-2/GFP (FIG. 8 (c)). In addition, greenfluorescence was not observed in the experiment using mock performedsimultaneously (FIG. 8 (a)). In addition, in either experiment, the redfluorescence of mCherry origin showed the equivalent coloring level.

SEQUENCE LISTING FREE TEXT

[SEQ ID NO: 1]

This shows the nucleotide sequence of cDNA of OATP1B3/ct.

[SEQ ID NO: 2]

This shows the nucleotide sequence of cDNA of OATP1B3/wt.

[SEQ ID NO: 3]

This shows the amino acid sequence of the protein which is coded byOATP1B3/wt.

[SEQ ID NO: 4]

This shows the nucleotide sequence of the frame 1-1 (including stopcodon) of OATP1B3/ct.

[SEQ ID NO: 5]

This shows the amino acid sequence of the peptide (f1-1) which is codedby the frame 1-1 of OATP1B3/ct.

[SEQ ID NO: 6]

This shows the nucleotide sequence of the frame 1-2 (including stopcodon) of OATP1B3/ct.

[SEQ ID NO: 7]

This shows the amino acid sequence of the peptide (f1-2) which is codedby the frame 1-2 of OATP1B3/ct.

[SEQ ID NO: 8]

This shows the nucleotide sequence of the frame 2 (including stop codon)of OATP1B3/ct.

[SEQ ID NO: 9]

This shows the amino acid sequence of the peptide (f2) which is coded bythe frame 2 of OATP1B3/ct.

[SEQ ID NO: 10]

This shows the nucleotide sequence of the frame 3 (including stop codon)of OATP1B3/ct.

[SEQ ID NO: 11]

This shows the amino acid sequence of the peptide (f3) which is coded bythe frame 3 of OATP1B3/ct.

[SEQ ID NO: 12]

This shows the nucleotide sequence of the forward primer used foramplification of a human GAPDH gene.

[SEQ ID NO: 13]

This shows the nucleotide sequence of the reverse primer used foramplification of a human GAPDH gene.

[SEQ ID NO: 14]

This shows the nucleotide sequence of the reverse primer used for theTouch-down PCR in RLM-5′-RACE.

[SEQ ID NO: 15]

This shows the nucleotide sequence of the reverse primer used for theNested-PCR in RLM-5′-RACE.

[SEQ ID NO: 16]

This shows the nucleotide sequence of the forward primer used for theRT-PCR and the cDNA cloning of OATP1B3/ct.

[SEQ ID NO: 17]

This shows the nucleotide sequence of the reverse primer used for theRT-PCR.

[SEQ ID NO: 18]

This shows the nucleotide sequence of the reverse primer used for thecDNA cloning of OATP1B3/ct.

[SEQ ID NO: 19]

This shows the nucleotide sequence of the forward primer used for thecDNA cloning of OATP1B3/wt.

[SEQ ID NO: 20]

This shows the nucleotide sequence of the reverse primer used for thecDNA cloning of OATP1B3/wt.

[SEQ ID NO: 21]

This shows the nucleotide sequence of the forward primer used for thedetection of OATP1B3/ct in the quantitative real-time PCR.

[SEQ ID NO: 22]

This shows the nucleotide sequence of the reverse primer used for thedetection of OATP1B3/ct in the quantitative real-time PCR.

[SEQ ID NO: 23]

This shows the nucleotide sequence of the forward primer used for thedetection of OATP1B3/wt in the quantitative real-time PCR.

[SEQ ID NO: 24]

This shows the nucleotide sequence of the reverse primer used for thedetection of OATP1B3/wt in the quantitative real-time PCR.

[SEQ ID NO: 25]

This shows the nucleotide sequence of the forward primer used for theexpression of the peptide consisting of the amino acid sequence fromf1-1/GFP/pcDNA3.1 of SEQ ID NO: 5.

[SEQ ID NO: 26]

This shows the nucleotide sequence of the reverse primer used for theexpression of the peptide consisting of amino acid sequence fromf1-1/GFP/pcDNA3.1 of SEQ ID NO: 5.

[SEQ ID NO: 27]

This shows the nucleotide sequence of the forward primer used for theexpression of a peptide consisting of the amino acid sequence fromf1-2/GFP/pcDNA3.1 of SEQ ID NO: 7, for the expression of a proteinconsisting of the amino acid sequence from f2/GFP/pcDNA3.1 of SEQ ID NO:9, and for the expression of a peptide consisting of the amino acidsequence from f3/GFP/pcDNA3.1 of SEQ ID NO: 11.

[SEQ ID NO: 28]

This shows the nucleotide sequence of the reverse primer used for theexpression of a peptide consisting of the amino acid sequence fromf1-2/GFP/pcDNA3.1 of SEQ ID NO: 7.

[SEQ ID NO: 29]

This shows the nucleotide sequence of the reverse primer used for theexpression of a protein consisting of the amino acid sequence fromf2/GFP/pcDNA3.1 of SEQ ID NO: 9.

[SEQ ID NO: 30]

This shows the nucleotide sequence of the reverse primer used for theexpression of a peptide consisting of the amino acid sequence fromf3/GFP/pcDNA3.1 of SEQ ID NO: 11.

[SEQ ID NO: 31]

This shows the nucleotide sequence of the forward primer used for theexpression of GFP protein from f1-1/GFP/pcDNA3.1, f1-2/GFP/pcDNA3.1,f2/GFP/pcDNA3.1, and f3/GFP/pcDNA3.1.

[SEQ ID NO: 32]

This shows the nucleotide sequence of the reverse primer used for theexpression of GFP protein from f1-1/GFP/pcDNA3.1, f1-2/GFP/pcDNA3.1,f2/GFP/pcDNA3.1, and f3/GFP/pcDNA3.1.

What is claimed is:
 1. An isolated deoxyribonucleic acid satisfying thefollowing conditions: (1) the isolated deoxyribonucleic acid is capableof hybridizing to a nucleic acid comprising the nucleotide sequence ofSEQ ID NO: 1 under a stringent condition; (2) the isolateddeoxyribonucleic acid does not hybridize to a nucleic acid comprisingthe nucleotide sequence of SEQ ID NO: 2 under the stringent condition orall but its 3′ terminus hybridizes to the nucleic acid comprising thenucleotide sequence of SEQ ID NO: 2 under the stringent condition; and(3) 20% to 80% of the length of the isolated deoxyribonucleic acidhybridizes to exon SV in SEQ ID NO: 1 under the stringent condition andthe length of the isolated deoxyribonucleic acid that does not hybridizeto exon SV under the stringent condition hybridizes to exon 3 of SEQ IDNO: 1 under the stringent condition, wherein the isolateddeoxyribonucleic acid includes a fluorescent label, a biotin label, aradioactive label, or an enzyme label and is 25 to 35 nucleotides inlength.
 2. A kit for measuring an alternative splicing variant ofOATP1B3 mRNA, comprising the isolated deoxyribonucleic acid according toclaim
 1. 3. An isolated deoxyribonucleic acid having the nucleotidesequence of SEQ ID NO: 1.